In vitro cytosine methylation of BRD7 promoter silence its activity in NPC cells. (A) Detection of the methylation effects of Met-pGL3-404/+46 and Met-pGL3-404/+46/GFP by restriction enzyme cutting of HpaII. pGL3-404/+46 was used as a control. (B) Detection of the luciferase activity of methylated BRD7 promoter construct Met-pGL3/-404,+46 in COS7, BHK-21, HNE1, CNE1, 6–10B, 5–8F, SW480, and Hela cells. Luciferase activity in COS7 and 5–8F cells is represented by black and gray histograms, respectively. All of the constructs were cotransfected with the SV40 β-galactosidase vector for normalizing transfection efficiency. Data are the means ± S.D. of three independent experiments. (C) Detection of the luciferase activity of modified GFP reporter construct Met-pGL3/-404,+46/EGFP in 5–8F cells. The full-length modified promoter construct pGL3/-404,+46/EGFP was used as a positive control. 38 h after transfection, the signal of EGFP fluorescence driven by promoter fragment -404/+46 or -266/-212 was observed by using an AX-80 analytical microscope system (Olympus, Tokyo, Japan).